(CANCER RESEARCH 50, 1470-1478. March I, 1990] Methyl /7-Hydroxyphenyllactate and Nuclear Type II Binding Sites in Malignant Cells: Metabolic Fate and Mammary Tumor Growth1 Barry M. Markaverich,2 Rebecca R. Gregory, MaryAnn Alejandro, Francis S. Kittrell, Daniel Medina, James H. Clark, Manju V'arma, and Rajender S. V'arma The Center for Biotechnology, Baylor College of Medicine, The Woodlands, Texas 77381 [B. M. M., R. R. G., M. A., M. V., K. S. V.], and Department of Cell Biology, Baylor College of Medicine, Houston. Texas 77030 [B. M. M., F. S. K., D. M., J. H. C.J ABSTRACT Previous studies in our laboratory demonstrated that methyl p-hydrox- yphenyllactate (MeHPLA) is an important cell growth-regulating agent which binds to nuclear type II binding sites in normal and malignant cells. Furthermore, this compound is deficient in a variety of rat and mouse mammary tumors and human breast cancer preparations, and this deficiency correlates with the loss of regulatory control. The present studies were performed to examine the metabolic fate of [3H)MeHPLA in mouse mammary tumors. Stable analogs of this compound such as 4,4'-dihydroxy benzylidene acetophenone were also assessed for nuclear type II site binding affinity and their ability to inhibit mammary cancer cell growth and proliferation in vitro and in vivo. The results demonstrate that mouse mammary tumors contain esterase activity which hydrolyzes MeHPLA to p-hydroxyphenyllactic acid, and this was the only major metabolite detected in these tumor preparations in vitro or in vivo. 4,4'- Dihydroxy benzylidene acetophenone, an esterase-stable MeHPLA an alog, was found to bind with high affinity to nuclear type II sites but not the estrogen receptor, was capable of occupying type II sites in cultured MCF-7 cells, and inhibited the proliferation of these cells in concentra tions which directly correlated with type II binding site occupancy. Similarly, 4,4'-dih>dro\y benzylidene acetophenone administration by silastic implant or injection resulted in a dose-dependent inhibition of the growth of transplantable mammary tumors in mice, suggesting that this stable analog mimicks MeHPLA as a cell growth-regulating agent. Taken together, these results suggest esterase hydrolysis of MeHPLA in mam mary tumors may result in a deficiency in this compound which correlates with a loss of regulatory control. INTRODUCTION MeHPLA3 is an endogenous ligand for nuclear type II sites which binds with high affinity (Kd ~ 5 HM)and inhibits normal and malignant cell growth and proliferation in vivo and in vitro (1-3). This compound is present in all normal tissues, which is consistent with its derivation from bioflavonoid (4) or tyrosine metabolism (5). Previous studies have demonstrated that a variety of rodent mammary tumors and human breast cancer preparations are deficient in MeHPLA, even though these tissues contained significant quantities of its precursor, HPLA (1-3). Unlike MeHPLA, HPLA does not bind to nuclear type II binding sites with high affinity (Kd ~200 nivi) and does not inhibit normal or malignant cell growth and proliferation in vivo or in vitro (2). Therefore, the deficiency of MeHPLA in tumors is consistent with constituitively high levels of type II Received 3/24/89; revised 9/18/89. 11/13/89; accepted 11/27/89. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 'Supported by research grants from NIH (CA-35480, CA-11944, and HD- 08436), the American Cancer Society (RD-266 and CH 469), the American Institute for Cancer Research (86B14 and R88B). and the State of Texas Advanced Biotechnology Program. 2To whom requests for reprints should be addressed. The Center for Biotech nology. Baylor College of Medicine, 4000 Research Forest Drive, The Woodlands, Texas 77381. J The abbreviations used are: MeHPLA, methyl p-hydroxyphenyllactate; HPLA, p-hydroxyphenyllactic acid; DMSO. dimethyl sulfoxide; DHBA, 4,4'- dihydroxy benzylidene acetophenone; DMEM, Dulbecco's modified Eagle me dium; DES, diethylstilbesterol; HPLC, high pressure liquid chromatography; FAA, flavone acetic acid; TE buffer, 10 mivi Tris, 1.5 mM EDTA, pH 7.4. sites which are measured in malignant tissues (1-3, 6-10). Since tumors contain HPLA, we reasoned that malignant cells metabolize MeHPLA and this is the basis for the deficiency in this important cell growth-regulating agent. The present studies were performed to define the metabolic fate of [3H]MeHPLA in malignant tissues and to determine whether stable analogs of this compound will inhibit malignant cell growth and proliferation in vivo and in vitro. These results suggest that malignant tissues contain an esterase(s) which hydrolyzes [3H]MeHPLA and that [3H]HPLA is the major metabolite in mouse mammary tumor preparations. A stable analog of MeHPLA, DHBA, which is not susceptible to ester ase hydrolysis, interacted with nuclear type II binding sites with high affinity and inhibited MCF-7 cell proliferation in vitro and the growth of mouse mammary tumors in vivo. These findings suggest that esterase activity in mammary tumors may be directly responsible for the deficiency of MeHPLA in these tissues, and this deficiency may be in part responsible for the lack of cell regulatory control associated with malignant cell proliferation. MATERIALS AND METHODS Animals and Treatment. Syngeneic BALB/c female mice bearing transplantable (8-10) mammary tumors (T-1504 and T-364 L; gener ations 2-10) were housed under controlled conditions (12 h light/day, 6:00 a.m. to 6:00 p.m.). Food and water were provided ad libitum. These tumors are moderately differentiated mammary ductal adenocar- cinomas which arose from a mammary preneoplastic outgrowth line D1M-36. When the tumors were 0.25-0.5 cm in size, the animals were randomly placed into groups (six animals/group) and treated with luteolin or DHBA as described in the text and figure legends. Luteolin and DHBA were injected in a DMSO/sesame oil (20% DMSO) or 0.9% saline vehicle or administered by silastic implant. The implants were prepared by sealing one end of the capsule with silastic cement and pipetting the test compound (dissolved in 100 n\ DMSO) into the open end. The capsules were sealed with silastic cement and allowed to cure 24 h prior to surgical implantation in the nape of the neck, under ether anesthesia. Controls were implanted with capsules containing 100 /¿I of DMSO vehicle. Tumors were measured (length x width) at the indicated times following treatment, and the results (mean tumor size) were expressed as a function of the day zero value. Tumors for bio chemical studies (binding assays and esterase assays) were removed from control or treated animals at the time indicated in the figure legends. Results were analyzed statistically utilizing analysis of variance and Duncan's new multiple range test (11). Chemicals and Reagents. MeHPLA was synthesized in our laboratory by treatment of HPLA with diazomethane, as previously described (2), and tritiated to a high specific activity (58 Ci/mmol) by Amersham Radiochemicals. Luteolin was purchased from Sarsynthese (Merignac, France). DHBA was synthesized by the condensation of equimolar quantities of/>-hydroxyacetophenone and/vhydroxybenzaldehyde (Aid- rich, Milwaukee, WI) under acidic conditions. The reaction product was purified by column chromatography on silica gel and DHBA was recrystallized from hexane/ether. The purity and structural confirma tion of these compounds were determined by high performance liquid chromatography, combined gas chromatography-mass spectrometry 1470 Research. on October 21, 2015. © 1990 American Association for Cancer cancerres.aacrjournals.org Downloaded from